1. Field of the Invention
This invention relates to non-proteinaceous organic molecules that exhibit the catalytic and kinetic properties of enzymes. More particularly, this invention relates to cyclodextrin-coenzyme and cyclodextrin-cofactor conjugates that behave catalytically and kinetically as do oxidation-reduction ("redox") enzymes.
2. Description of the Related Art
Enzymes are proteins with catalytic activity that exhibit high specificity and large rate accelerations. Although enzymes are large and complex molecules, their power to catalyze chemical reactions can be attributed mainly to binding of reactants and catalysis. Binding not only is largely responsible for the specificity of the reaction but, by stereochemistry, also brings the substrate in close proximity to and in the correct orientation with the active site(s) of the enzyme. Other factors, such as the microenvironment of the catalytic site and the stabilization of the transition state by hydrogen bonding, contribute to enzyme activity, but binding (seen particularly in transition-state analogues) and catalysis are the two essential features of all enzymes. See, for a review of enzymes, Dixon, M., and Webb, E. C., Enzymes, Academic Press, N.Y., 1979.
An additional substance besides the enzyme and substrate is required in many cases in order that the reaction may proceed. Although such substances, variously referred to as `cofactors` or `coenzymes` may participate in the intermediate steps of the reaction catalyzed by the enzyme (or the cycle of reactions catalyzed by a system of enzymes), they are not consumed during the process, but are found in their original form at the end of the catalysis. They may, therefore, be regarded as an essential part of the catalytic mechanism.
The majority of coenzymes and cofactors act in one of the following ways: (a) as inter-enzyme carriers; (b) as a prosthetic group, which often is an intraenzyme carrier (e.g., heme, flavin, nicotinamide, pteridines, coenzyme Q, a metal atom or ion, etc.) covalently or electrostatically bound to the enzyme protein as an essential part of the enzyme; (c) by altering the shape of the enzyme molecule; (d) by subunit aggregation; (e) as stabilizers; (f) as templates; (g) as primers; and (h) as intermediates.
Enzymes are labile molecules, and this lability limits their industrial usefulness. They are sensitive to heat and pressures which, in the extreme, can reduce or destroy catalytic activity and, in the further extreme, can denature and precipitate the enzyme protein. Many enzymes are also sensitive to extremes of pH which can irreversibly inactivate the enzyme. The presence of proteolytic enzymes, whether of bacterial or other origins, will also reduce or destroy the effectiveness of enzymes. Certain heavy metal ions may also inactivate enzymes. For these reasons, non-protein artificial enzymes that are not subject to these problems have been sought for many years.
Non-protein artificial enzymes, also referred to as miniature organic models of enzymes, have been known since about 1970 when Breslow et al. disclosed an `artificial enzyme combining a metal catalytic group and a hydrophobic cavity`. Breslow, R., et al., J. Am. Chem. Soc. 92:1075 (1970). See also, for a review: Breslow, R., Cold Sorinq Harbor Symposium on Quantitative Biology, 52:75-81 (1987).
As noted above, enzymes operate by binding a substrate and then performing a selective catalyzed reaction within the enzyme-substrate complex. The geometry of the complex and the geometric placement of various catalytic functional groups help determine both the rates and the specificities of the reaction. Among artificial enzymes, a generally useful type of binding appears to be hydrophobic inclusion within a cavity. Breslow et al., 1970 above, showed how hydrophobic binding in a cavity could be used to bring a simple organic compound close enough to a metal to permit metal-catalyzed reactions, even though the substrate itself is not a normal metal ligand.
The ideal artificial enzyme should not only have a cavity that provides maximum hydrophobic interaction with a substrate to form complexes, but the cavity should fit bulky components of the substrate such as aromatic rings, and orient the functional group of the bound substrate toward the attacking atom or group. D'Souza, V. T. et al., Acc. Chem. Res., 20:146-152 (1987).
The cyclodextrins are cyclic molecules with a relatively hydrophobic interior cavity and hydroxyl groups that make them water soluble. Bender, M. L., et al., Cyclodextrin Chemistry, Springer-Verlag, N.Y. 1977; Tabushi, I., Acc. Chem. Res., 15:66 (1982). Cyclodextrins consisting of 6 (.alpha.-cyclodextrin or cyclohexaamylose), 7 (.beta.-cyclodextrin or cycloheptoamylose) and 8 (.gamma.-cyclodextrin or cyclooctaamylose) units of .alpha.-1,4-linked D-glucopyranoses are known. Cyclodextrins have doughnut shapes with secondary hydroxyl groups at the C-2 and C-3 atoms of glucose units disposed in the more open end and primary hydroxyl groups at the C-6 atom of the glucose unit located at the other end (1). ##STR1## where n=6, 7 or 8. The interior of the cavity, consisting of a ring of C--H groups, a ring of glycosidic oxygen atoms, and another ring of C--H groups, is hydrophobic in nature. The inner diameters of the cavities are approximately 4.5 .ANG. in .alpha.-cyclodextrin, 7.0 .ANG. in .beta.-cyclodextrin and 8.5 .ANG. in .gamma.-cyclodextrin. D'Souza et al., 1987, above. .alpha.- and .beta.-Cyclodextrins would provide a snug fit for an aromatic ring. Formation of inclusion complexes with various substrates (binding) is one of the most important characteristics of cyclodextrins. Bender, M. L., et al., Adv. Enzymol. Relat. Areas Mol. Biol., 58:1 (1986).
Breslow et al. (Breslow, R., et al., J. Am. Chem. Soc., 105:1390-1391 (1983)), have produced a `synthetic transaminase` enzyme wherein the coenzyme pyridoxamine is linked to the C-6 of .beta.-cyclodextrin, thereby putting it on the more narrow primary end of the structure. The artificial enzyme is reportedly able to transaminate keto acids, with a preference for keto acids containing an hydrophobic aromatic group, e.g., phenylpyrunic acid. The coenzyme was also attached to the secondary face of the molecule via the C-3 hydroxyl group, but, although this molecule reportedly also catalyzes transamination and also prefers aromatic keto acids as substrates, the secondary-side derivative is only about half as effective as is the primary side analogue. Further, the primary-side derivative gives a preference for the synthesis of the natural (in vertebrates) L-enantiomers of amino acids, whereas the secondary-side derivatives give a preference for the synthesis of the unnatural D-product.
D'Souza et al. (D'Souza, V. T., et al., Biochem. Biophys. Res. Commun., 129:725 (1985)) have synthesized a `synthetic chymotrypsin` proteolytic enzyme wherein .alpha., .beta. and .gamma.-cyclodextrins are functionalized by derivatization at the secondary-side 2-hydroxyl group with o- [4(5)-mercaptomethyl-4(5)-methylimidazol-2-yl] benzoic acid, to produce a derivative designed to mimic the active site of chymotrypsin itself. The artificial and natural enzymes are reportedly comparable in their catalytic activity. Further, whereas the real chymotrypsin has an optimal temperature around 45.degree. C., it precipitates after about 55.degree. C. and is rendered inactive; in contrast, the activity of the artificial enzyme keeps increasing to at least 80.degree. C.
Breslow et al. (Breslow, R., et al., J. Am. Chem. Soc., 100:3225 (1978)) constructed .beta.-cyclodextrinyl bisimidazole which is a model for the ribonuclease enzyme. In this artificial enzyme, the bisimidazole is linked to two of the primary-side 6-hydroxyl groups of the .beta.-cyclodextrin, forming a bifunctional catalytic site. The artificial enzyme was reportedly slow compared to ribonuclease itself, although exhibiting characteristics of the enzyme.
The class of protein enzymes referred to variously as oxidoreductases or redox enzymes includes enzymes concerned with biological oxidation and reduction, and therefore with respiration, fermentation, and metabolism in general. Oxidoreductases include (a) dehydrogenases and oxidases that employ, e.g., AND, NADP, FMN and electron-transferring flavoproteins, as coenzymes; (b) peroxidases that can be iron-containing heme proteins or flavoproteins; and (c) oxygenases or hydroxylases that can be flavoproteins, use pteridines or 2-oxoglutamate as coenzymes, or use copper ions as an oxidation / reduction pair. Such enzymes have great medical and industrial potential. However, such applications are limited by the instability of these enzymes to high temperatures and pressures, mechanical stress, organic solvents and detergent conditions. Thus, artificial redox enzymes that carry out the catalytic functions of natural redox enzymes but that are stable to the conditions noted above, would be extremely valuable.
Limited success has been achieved with an artificial flavoenzyme. Tabushi et al. (Tabushi, I., et al., J. Am. Chem. Soc., 109:4734-4735 (1987)) reported the synthesis of an artificial flavoenzyme, flavo-.alpha.-cyclodextrin in which the 8-position of the flavin is attached to the primary-side 6-position. This molecule reportedly carries out electron transport, although it was also disclosed in this report that the natural NADH-dependent flavoprotein enzyme exhibits a rate constant 30-fold greater than that of the artificial enzyme, and the natural flavoprotein exhibits an association constant for NADH 8-fold greater than does the artificial enzyme. In addition, the reported chemical synthesis of this artificial is difficult, as the riboflavin decomposes under the mildly basic conditions that are required to attach the coenzyme to the 6-position of cyclodextrin by the disclosed process (see below in Detailed Description of the Invention).
Thus, a great need exists for highly active and stable, readily and inexpensively synthesizable artificial redox enzymes. This need has been fulfilled by the invention disclosed below.